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Department of Animal Sciences
The Robert H. Smith Faculty
of Agricultural, Food & Environment

The Hebrew University of Jerusalem.

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Petersen, E. ; Mills, E. ; Miller, S. I. Cyclic-di-GMP regulation promotes survival of a slow-replicating subpopulation of intracellular Salmonella Typhimurium. Proceedings of the National Academy of Sciences 2019, 116, 6335. Publisher's VersionAbstract
Cyclic di-GMP is a bacterial second messenger that transmits extracellular signals to the intracellular environment via sensor cyclic-di-GMP−metabolizing enzymes. Here a fluorescent biosensor is used to accurately measure cyclic-di-GMP concentrations in thousands of individual intracellular Salmonella Typhimurium. Furthermore, three enzymes that reduce cyclic-di-GMP concentrations were identified and shown to be essential for reduction of cyclic di-GMP, intracellular survival, and full virulence for mice. This was due to cyclic-di-GMP−mediated overproduction of cellulose that specifically affected a population of slowly replicating bacteria. These results further our knowledge of mechanisms of virulence and persistence of this important pathogen.Salmonella Typhimurium can invade and survive within macrophages where the bacterium encounters a range of host environmental conditions. Like many bacteria, S. Typhimurium rapidly responds to changing environments by the use of second messengers such as cyclic di-GMP (c-di-GMP). Here, we generate a fluorescent biosensor to measure c-di-GMP concentrations in thousands of individual bacteria during macrophage infection and to define the sensor enzymes important to c-di-GMP regulation. Three sensor phosphodiesterases were identified as critical to maintaining low c-di-GMP concentrations generated after initial phagocytosis by macrophages. Maintenance of low c-di-GMP concentrations by these phosphodiesterases was required to promote survival within macrophages and virulence for mice. Attenuation of S. Typhimurium virulence was due to overproduction of c-di-GMP−regulated cellulose, as deletion of the cellulose synthase machinery restored virulence to a strain lacking enzymatic activity of the three phosphodiesterases. We further identified that the cellulose-mediated reduction in survival was constrained to a slow-replicating persister population of S. Typhimurium induced within the macrophage intracellular environment. As utilization of glucose has been shown to be required for S. Typhimurium macrophage survival, one possible hypothesis is that this persister population requires the glucose redirected to the synthesis of cellulose to maintain a slow-replicating, metabolically active state.
Katsowich, N. ; Elbaz, N. ; Pal, R. R. ; Mills, E. ; Kobi, S. ; Kahan, T. ; Rosenshine, I. Host cell attachment elicits posttranscriptional regulation in infecting enteropathogenic bacteria. Science 2017, 355, 735–739. Publisher's VersionAbstract
Escherichia coli is transformed from a commensal organism into a pathogen by acquisition of genetic elements called pathogenicity islands (PAIs). Katsowich et al. investigated how the PAI virulence genes of enteropathogenic E. coli (EPEC) respond when the bacterium attaches to a host gut cell. EPEC first sticks to the host by means of pili and then uses a PAI-encoded type 3 secretion system (T3SS) to inject multiple effectors into the host cell. But not all virulence mediators are injected. For example, CesT, a bacterial chaperone, delivers virulence effectors into the T3SS apparatus. Then, within the bacterial cytoplasm, it interacts with a gene repressor called CsrA, which reprograms bacterial gene expression to help the bacteria to adapt to epithelial cell–associated life.Science, this issue p. 735The mechanisms by which pathogens sense the host and respond by remodeling gene expression are poorly understood. Enteropathogenic Escherichia coli (EPEC), the cause of severe intestinal infection, employs a type III secretion system (T3SS) to inject effector proteins into intestinal epithelial cells. These effectors subvert host cell processes to promote bacterial colonization. We show that the T3SS also functions to sense the host cell and to trigger in response posttranscriptional remodeling of gene expression in the bacteria. We further show that upon effector injection, the effector-bound chaperone (CesT), which remains in the EPEC cytoplasm, antagonizes the posttranscriptional regulator CsrA. The CesT-CsrA interaction provokes reprogramming of expression of virulence and metabolic genes. This regulation is likely required for the pathogen’s adaptation to life on the epithelium surface.
Mills, E. ; Avraham, R. Breaking the population barrier by single cell analysis: one host against one pathogen. Current Opinion in Microbiology 2017, 36, 69 - 75. Publisher's VersionAbstract
Most of our understanding of the host–bacterium interaction has come from studies of bulk populations. In reality, highly adaptable and dynamic host cells and bacteria engage in complex, diverse interactions. This complexity necessarily limits the depth of understanding that can be gained with bulk population measurements. Here, we will review the merit of single cell analysis to characterize this diversity that can trigger heterogeneous outcomes. We will discuss heterogeneity of bacterial and host populations, differences in host microenvironments, technological advances that facilitate the analysis of rare subpopulations, and the potential relevance of these subpopulations to infection outcomes. We focus our discussion on intracellular bacterial pathogens and on methods that characterize and quantify RNA in single cells, aiming to highlight how novel methodologies have the potential to characterize the multidimensional process of infection and to provide answers to some of the most fundamental questions in the field.